The objective is to create a global gene expression atlas of craniofacial development. The central thesis is that a combination of laser capture microdissection, and FACS, combined with microarrays can be used to efficiently achieve this goal. Microarrays with essentially complete gene representation can be used to rapidly determine the expression levels of every gene in laser capture microdissected elements of craniofacial development. A single experiment, therefore, provides a comprehensive analysis of the gene expression status of one component and a limited number of experiments examining each structure and cell type can create an atlas. A combination of structure, lectin staining, and transgenic GFP expression will be used to precisely identify specific compartments and lineages, including cells driving neural crest induction, nasal placodes and pits, lateral and medial facial eminences, neural crest and paraxial mesoderm cells, maxilla and mandibular recesses, signaling centers, and the structures of palatogenesis. Specific Aim 2, more limited in scope, is to make Sp8-GFP-Cre and Fgf8-GFP-Cre transgenic mouse tools, which will serve a dual purpose, to allow identification of additional discrete craniofacial components for Specific Aims 1 and 3, and to aid future domain specific gene knockout studies by the craniofacial research community. Specific Aim 3 is to characterize the Sp8 mutant mouse, which shows a massive disruption of craniofacial development. We hypothesize that Sp8 is upstream of FGF signaling first in the medial neuroepithelium of the forebrain, important for driving neural crest proliferation and migration, and later in the nasal placodes and pits, important for signaling the forming facial eminences. We propose a combined molecular marker analysis;a Cre based genetic dissection of Sp8 function, and a microarray based gene expression profile comparison of signaling centers in wild type and Sp8 mutants. PUBLIC HEALTH RELEVANCE: Cleft lip and palate are very common birth defects, and there are many other facial malformations that are more severe, but less common. These problems have both genetic and environmental causes. We propose to use the latest technologies to study all of the genes involved in making the face, to gain a better understanding of this complicated process. It is hoped this work will lead to new methods to prevent and/or treat these birth defects.